Aging research focuses the molecular mechanisms of age-related diseases and metabolic deregulation is now appreciated to be associated with and causal in several human diseases of aging. However, the mechanisms of how altered metabolism leads to age-related diseases are not known. Sirtuin regulation of mitochondrial protein modifications controls several aspects of metabolism and is emerging as important regulatory node in aging. The goal of this project is to understand how protein modifications are regulated by the NAD(+)-dependent sirtuin deacylase SIRT4, and how this sirtuin influence the diseases of aging. While all the other sirtuins have a robust enzymatic activity in vitro, SIRT4 so far has remained enigmatic. Recently, we combined phylogenetic, structural, biochemical, and physiological data and identified two novel enzymatic activities of SIRT4. This information provides a deeper understanding of metabolic regulation and the aging process. The overall goal of this proposal is to understand the role of SIRT4 in aging; three specific aims will be pursued: 1. Determine the changes in two novel protein acyl modifications, using an innovative quantitative proteomic strategy; 2. Determine the effect of these acyl modifications on protein function by using a combination of cell and murine models, in order to measure the changes in protein activity of specific proteins, as well as the overall function of mitochondria; 3. Determin role of SIRT4 on aging by measuring several physiological parameters of aging using both in vitro cellular assays and in vivo assays in mice, with the overall goal to determine how SIRT4 maintains mitochondrial and cellular homeostasis during aging. This study combines a set of novel protein modifications, a comprehensive experimental design, and an innovative conceptual framework. Furthermore, this study will build a foundation of knowledge to further understand the cellular mechanisms of aging and disease. Importantly, the proposed research is significant to the field of aging because it is expected to advance and expand understanding how metabolic deregulation influences aging, and how this contributes to age-related diseases. Ultimately, such knowledge has the potential to inform the development of new therapies against diseases of aging and the aging process itself.